Paramyxoviruses infect the respiratory tract and induce a high level of local and systemic immunity. Reverse genetics systems for producing recombinant viruses have been developed in our laboratory and by collaborators, providing the means for introducing foreign genes to create vectored vaccines. This type of virus has a negligible incidence of recombination, obviating concerns of genetic exchange with circulating viruses. The respiratory tropism provides for direct immunization of the respiratory tract, an advantage against pathogens that use the respiratory tract as a portal for entry and egress. The particular paramyxoviruses that we are evaluating replicate in the superficial cells of the respiratory tract and do not spread significantly beyond that site, reducing concerns about infection of distal tissues. Thus, paramyxoviruses have distinct advantages as vaccine vectors for expressing protective antigens of highly pathogenic agents. We have been evaluating HPIV3 as a human viral model, and avian Newcastle disease virus (NDV) as a non-human viral model. [unreadable] We previously used a derivative of HPIV3 as a vector to express the structural proteins of the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV). This identified the spike S glycoprotein as the only significant neutralization and protective antigen among the SARS structural proteins. A PIV3-vectored vaccine expressing S was immunogenic and protective against SARS-CoV in African green monkeys.[unreadable] We also previously evaluated HPIV3 as a vector to express the single glycoprotein GP of Ebola virus (EBOV). Guinea pigs infected with a single intranasal inoculation of HPIV3/EboGP developed a strong serum antibody response and were completely protected against disease associated with challenge with an otherwise highly lethal intraperitoneal injection of EBOV. A single inoculation of the respiratory tract of rhesus monkeys was moderately immunogenic against EBOV and protected 88% of the animals against severe hemorrhagic fever and death caused by EBOV challenge. Two doses were highly immunogenic and all of the animals were free of disease signs and detectable EBOV challenge virus.[unreadable] The vaccines described above are candidates for further evaluation as pediatric vaccines. However, it was unclear whether they would be effective in adults because the high seroprevalence against HPIV3 in that population might restrict the replication and immunogenicity of an HPIV3 vector. To evaluate this possibility, guinea pigs were infected with HPIV3 and, 40 days later, were immunized by infection with HPIV3/EboGP. Replication of HPIV3/EboGP was not detected in any of the animals, indicating that immunity to HPIV3 indeed strongly restricted the replication of the vector. Surprisingly, however, the immune response to EBOV GP was almost equivalent to that achieved in control animals that had not been previously infected with HPIV3. Thus, it may be possible to achieve immunization in HPIV3-immune animals. We are presently evaluating the immunogenicity of this vaccine in HPIV3-seropositive primates.[unreadable] Next, we deleted the F and HN genes from HPIV3 and replaced them with EBOV GP to create a virus, HPIV3/&#8710;F-HN/EboGP, in which GP would be the sole viral transmembrane surface protein. This virus was attenuated in vitro but reached titers comparable to those of HPIV3. Electron microscopy showed that the shape and dimensions of the HPIV3/&#8710;F-HN/EboGP particle resembled those of HPIV3 (and HPIV3/EboGP) but the envelope thickness and spike appearance resembled those of EBOV. In an in vitro model of the human epithelium, HPIV3/&#8710;F-HN/EboGP was highly attenuated and displayed the apical infection phenotype of HPIV3 and did not spread beyond the superficial cell layer. Following intranasal infection of guinea pigs, HPIV3/&#8710;F-HN/EboGP was highly attenuated and completely restricted to the respiratory tract but nonetheless was highly immunogenic. A single intranasal dose provided complete protection of guinea pigs against an otherwise lethal challenge of guinea pig-adapted EBOV, such that there were no disease signs or evidence of challenge virus replication. Since HPIV3/&#8710;F-HN/EboGP lacks the two neutralization antigens of HPIV3, it should be even less sensitive to pre-existing HPIV3-specific immunity than HPIV3/EboGP. We confirmed that the virus was insensitive to neutralization by HPIV3-specific antibodies in vitro. In addition, there was no significant difference in its immunogenicity in guinea pigs that were HPIV3-nave versus HPIV3-experienced. Thus, HPIV3/&#8710;F-HN/EboGP provides an alternative to HPIV3/EboGP, one that is very highly attenuated, is insensitive to HPIV3-neutralizing antibodies, and nonetheless is nearly as immunogenic. [unreadable] A second strategy to overcome seroprevalence is to use an animal virus that does not usually infect humans and is antigenically distinct from common human viruses. Newcastle disease virus (NDV) is an avian paramyxovirus (comprising serotype 1) that fits these criteria. A non-human virus has the potential to be naturally attenuated in primates due to host range restriction, and this indeed appears to be the case with NDV. There is serological evidence that bird handlers can be infected, but infection apparently does not cause significant disease. We confirmed that NDV is very highly attenuated following inoculation of the respiratory tract of rhesus and African green monkeys. NDV exists naturally in a variety of strains that exhibit a wide spectrum of virulence in birds, ranging from highly virulent (velogenic), to moderate virulence (mesogenic), to low virulence (lentigenic). We have compared vectors based on lentigenic, mesogenic, and composite NDV strains and have found little difference in attenuation in primates, suggesting that the pathotype distinctions that are so prominent in birds are overshadowed in primates by the strong host range restriction. [unreadable] Despite the high level of attenuation, which would be predictive of a high level of vaccine safety, the expressed foreign proteins are moderately-to-highly immunogenic. For example, an NDV vector was constructed to express the SARS-CoV S protein. African green monkeys immunized via the respiratory tract with two doses of the vaccine developed a titer of SARS-CoV-neutralizing antibodies comparable to the robust secondary response observed in animals that have been immunized with a different experimental SARS-CoV vaccine and challenged with SARS-CoV. When animals immunized with NDV-S were challenged with a high dose of SARS-CoV, direct viral assay of lung tissues taken by necropsy at the peak of viral replication demonstrated a 236-1,102 fold mean reduction in pulmonary SARS-CoV titer compared to control animals.[unreadable] Another NDV was engineered to express the hemagglutinin HA glycoprotein of highly pathogenic avian H5N1 influenza virus (HPAIV) (NDV-HA). The NDV-HA virus was highly attenuated in African green monkeys as well as in chickens. In African green monkeys, two doses of NDV-HA induced a titer of HPAIV-neutralizing serum antibodies that, based on historic controls, would be consistent with substantial protection against morbidity and mortality caused by H5N1 HPAIV. Moreover, a substantial respiratory mucosal immunoglobulin A response was induced following one and two doses, which would be particularly important in controlling a respiratory pathogen. NDV has potential for further development as a highly attenuated vaccine vector for use in humans against highly pathogenic agents.[unreadable] We have initiated antigenic and sequence analysis of serotypes 2-9 of avian paramyxoviruses (APMV) as a prelude to their evaluation for attenuation and safety in non-human primates as potential vectors. Complete sequences have been determined for representatives of APMV2 and APMV3.